Structural insight into how RAD51 paralog exchange regulates RAD51 filament formation

Nature Structural & Molecular Biology · 2026-04-22 · 1 citations

Homologous recombination (HR) repairs DNA double-strand breaks and stabilizes stressed replication forks, and HR deficiency promotes genome instability and cancer. HR requires assembly of RAD51 nucleoprotein filaments on single-stranded DNA (ssDNA), a process regulated by the human RAD51 paralogs RAD51C, XRCC3, RAD51D and XRCC2. Here, using cryo-electron microscopy, we find that the RAD51-XRCC3-RAD51C complex (RAD51-X3C) assembles into an octamer in which XRCC3 engages the RAD51 DNA-binding surface and RAD51 subunits adopt a misaligned configuration incompatible with filament formation. These features define an autoinhibited RAD51-X3C state that limits nonproductive RAD51 binding to double-stranded DNA or RNA-DNA hybrids while preserving RAD51 availability for ssDNA-dependent strand exchange. We further show that the RAD51D-XRCC2 paralog complex remodels RAD51-X3C into a pentameric RAD51-X3CDX2 assembly by engaging the exposed RAD51C surface and disrupting contacts that stabilize the octamer. This remodeling exposes the RAD51 DNA-binding interface, enhances RAD51-ssDNA filament assembly, and promotes strand exchange on RPA-coated ssDNA, and yields a filament-compatible paralog assembly that integrates into ssDNA-bound RAD51 filaments. Together, these findings establish paralog exchange as a mechanism that converts an autoinhibited RAD51-X3C octamer into an activated RAD51-X3CDX2 pentamer to regulate RAD51 filament formation during HR and replication fork preservation.

Unveiling BCL-xL-specific PROTAC Efficiency and Dissociation Mechanisms Using Native Mass Spectrometry

ChemRxiv · 2025-09-23 · 1 citations

Overexpression of anti-apoptotic proteins such as BCL-xL is a hallmark of various cancers and a major driver of resistance to conventional chemotherapies. While small-molecule BCL-xL inhibitors have shown promising outcomes, their clinical use is hindered by dose-limiting toxicities, especially thrombocytopenia. Proteolysis-targeting chimeras (PROTACs) offer a promising alternative by promoting selective degradation of target proteins via the ubiquitin-proteasome system, thereby reducing off-target effects associated with small molecule inhibitors. However, rational design and optimization of PROTACs remain challenging due to the need to balance simultaneous interactions with both an E3 ubiquitin ligase and the target protein. Here we employ native mass spectrometry (MS) as a rapid, label-free platform to screen and characterize the formation and stability of ternary complexes between BCL-xL, VHL E3 ligase complex (VCB), and various targeting PROTACs. Native MS enables direct detection of binary BCL-xL●PROTAC and ternary BCL-xL●PROTAC●VCB complexes and provides semi-quantitative insights into PROTAC affinity and cooperativity with both binding partners. Furthermore, we explore the dissociation pathways of these complexes in the gas phase using collision-induced dissociation (CID) and ultraviolet photodissociation (UVPD), revealing distinct fragmentation and subunit release patterns that reflect the structural organization and gas-phase stability of the complexes. Variable-temperature ESI-MS (vT-ESI) further allows assessment of thermal stabilities of the complexes in solution. Together, our study demonstrates the power of native MS to both screen and mechanistically characterize PROTAC-induced ternary complex formation.

Targeting DDR2 for Treating Pancreatic Cancer

Molecular Cancer Therapeutics · 2025-06-26

Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer with limited effective treatments, partly because of its complex tumor microenvironment. In this study, we report discoidin domain receptor 2 (DDR2), a receptor tyrosine kinase, as a critical protein that promotes PDAC growth and survival. Our results reveal that DDR2 is highly expressed and its expression correlates with the worst survival outcome in patients with PDAC. Using an unbiased high-throughput screen of small-molecule inhibitor libraries, we identified CIDD-8633, a novel inhibitor targeting DDR2. Our study suggests that CIDD-8633 interacts with DDR2 and inhibits DDR2-associated signaling. Importantly, in vivo studies demonstrate that CIDD-8633 effectively blocks PDAC tumor growth in preclinical mouse models. Additionally, combining CIDD-8633 with gemcitabine enhanced its efficacy synergistically. Mechanistically, CIDD-8633 treatment induces pro-apoptotic genes in PDAC cells. These findings position DDR2 as a promising therapeutic target and CIDD-8633 as a potential DDR2 inhibitor, offering new avenues for the treatment of PDAC.

Single-Assay Characterization of Ternary Complex Assembly and Activity in Targeted Protein Degradation

bioRxiv (Cold Spring Harbor Laboratory) · 2025-08-22

Targeted protein degradation (TPD) is a rapidly advancing therapeutic strategy that selectively eliminates disease-associated proteins by co-opting the cell's protein degradation machinery. Covalent modification of proteins with ubiquitin is a critical event in TPD, yet the analytical tools for quantifying the ubiquitination kinetics have been limited. Here, we present a real-time, high-throughput fluorescent assay utilizing purified, FRET-active E2-Ub conjugates to monitor ubiquitin transfer. This assay is highly versatile, requiring no engineering of the target protein or ligase, thereby accelerating assay development and minimizing the risk of artifacts. The single-step, single-turnover nature of the monitored reaction enables rigorous and quantitative analysis of ubiquitination kinetics. We show that this assay can be used to measure key degrader characteristics such as degrader affinity for the target protein, degrader affinity for the ligase, affinity of ternary complex assembly, and catalytic efficiency of the ternary complex. The high sensitivity and accuracy of this comprehensive, single-assay approach to ternary complex characterization will empower the discovery and optimization of heterobifunctional degraders and molecular glues.

Abstract A025-PR001: The potential of BCL-xL degradation as a strategy to eliminate chemotherapy-resistant neuroblastoma persister cells

Cancer Research · 2025-09-25

Abstract BACKGROUND: We recently showed that tumor-intrinsic and -extrinsic activation of NFκB is a major mediator of high-risk neuroblastoma (HRNB) chemoresistance (Grossmann et al., Cancer Discovery, ‘24). The anti-apoptotic protein BCL-xL is a downstream effector of the NFκB pathway and is upregulated in HRNB persister cells after chemotherapy. While showing efficacy, BCL-xL small molecule inhibitor development has been limited by on-target off-tumor thrombocytopenia. DT-2216 (DT), a BCLxL PROteolysis TArgeting Chimera (PROTAC), mitigates this toxicity by exploiting the E3 ligase VHL, expressed at low levels in platelets. AIMS: To validate NFκΒ-mediated BCL-xL overexpression as a HRNB chemoresistance mechanism, and exploit this therapeutic vulnerability to eradicate persister cells using DT. METHODS: Isogenic BCL-xLhigh and BCL-xL low cell lines were generated using an overexpression vector and a doxycycline-inducible CRISPRi system. Six human HRNB cell lines were treated with topotecan/cyclophosphamide (topo/cpm) and DT. DC50 and IC50 values were determined via immunoblots and cell viability assays over 5 days. Five chemotherapeutics were combined with DT in 6x6 dose-response matrices. Zero Interaction Potency (ZIP) scores were calculated using R software. Two Patient-Derived Xenograft (PDX) HRNB flank models in CB17-SCID mice were treated with two subsequent cycles of topo/cpm +/- low-dose DT (15mg/kg) for 6 weeks. Three PDXs were treated with vehicle or escalating DT doses (15mg/kg, 22.5mg/kg) for 2 weeks. Intratumoral BCL-XL degradation was determined by immunoblots. Complete blood counts were measured weekly. RESULTS: BCL-xL overexpression and knock-down significantly (p<0.05) enhanced and reduced cell viability following chemotherapy, respectively. Combination of high-dose (>IC50) topo/cpm and 500nM-1μM DT facilitated the eradication of persister cells in vitro. DT demonstrated synergy with all tested chemotherapeutics across multiple cell lines. The ZIP scores were higher (ZIP>10) around the chemotherapy IC50 and DT DC50, indicating a clinically-relevant range-specific therapeutic synergy. However, DT monotherapy at 15mg/kg twice weekly showed incomplete degradation of BCL-xL at day 7 (55%, 37-70%) and 14 (79%, 43-112%) and no effect on tumor growth across three PDXs. Combining DT with topo/cpm in two PDXs did not lead to significant enhancement of chemotherapy anti-tumor efficacy. Escalating DT doses to 22.5 mg/kg led to further BCL-xL depletion at day 7 (44%, 27-61%) and day 14 (49%, 26-73%) across three models. Dose-dependent thrombocytopenia was the only observed side effect, with platelet counts (x10^5/uL) of 15 [10-18], 4 [2-10], 2 [1-4] in vehicle, 15mg/kg, 22.5mg/kg arms, respectively. CONCLUSIONS: BCL-xL mediates chemoresistance in HRNB. Combination of DT and chemotherapy is synergistic in vitro. DT showed incomplete BCL-xL degradation in vivo at the dose and schedule tested here, which may be required for anti-tumor efficacy. Ongoing efforts may provide insight into de novo or acquired mechanisms of DT resistance. Citation Format: Matteo Calafatti, Emily Mycek, Dana Al-halawani, Josh Kalna, David Groff, Esther Coronado Mondragón, Patel Khushbu, Mark Gerelus, Jarrett Lindsay, Kyabeth Torres-rodriguez, Gregory Connelly, Smita Matkar, Sajid Khan, Zhengyu Wang, Hong Yu Li, Jaime Font de Mora, Yael P Mossé, Daohong Zhou, Liron Grossmann, John M Maris. The potential of BCL-xL degradation as a strategy to eliminate chemotherapy-resistant neuroblastoma persister cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Discovery and Innovation in Pediatric Cancer— From Biology to Breakthrough Therapies; 2025 Sep 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_2):Abstract nr A025-PR001.

Machine Learning-Assisted Terahertz Metagrating Biosensor for Label-Free Bacterial Identification Based on Spectral Fingerprinting

Analytical Chemistry · 2025-10-02 · 2 citations

Rapid and accurate identification of pathogenic bacteria is pivotal for enabling early clinical diagnosis and precision antimicrobial therapy. In this study, we developed a label-free bacterial detection platform that integrates terahertz (THz) metagrating with machine learning algorithms to identify specific pathogens through spectral fingerprint analysis. This approach uses all-dielectric THz metagrating fabricated via 3D printing to amplify wave–analyte interactions, generating distinct resonance-enhanced spectral signatures. Supervised machine learning algorithms were employed to discern subtle spectral variations, in which support vector machines achieved high performance in identifying eight pathogens with an accuracy of 97.50%. Furthermore, our approach exhibited exceptional performance on two newly recruited clinical cohorts, achieving overall accuracies of 99.29% and 96.75%, with AUC values exceeding 0.99 for all eight bacterial species. Notably, 100% accuracy was achieved for Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, and Enterococcus faecium across all the data sets. Our platform achieves complete bacterial identification within 5 min postculture, offering the advantages of being simple, rapid, cost-effective (<$0.10 per test), and compatible with downstream analytical technologies, displaying robust potential as a universal tool for clinical microbial identification.

Discovery of Dual BCL-xL/BCL-w Degraders by Exploiting the Bis(sulfonyl)benzene Ring of ABT-263 as a Linkage Vector

Journal of Medicinal Chemistry · 2025-08-29 · 2 citations

Targeting antiapoptotic proteins BCL-xL, BCL-2, and BCL-w has been extensively investigated for cancer treatment. However, robust inhibition of BCL-xL by conventional inhibitors, such as ABT-263, causes thrombocytopenia, a notable drawback that limits the clinical utility of this strategy. To overcome this on-target toxicity, BCL-xL-selective and BCL-xL/BCL-2 dual-targeting proteolysis targeting chimeras (PROTACs) have been developed as alternative therapeutic strategies. In this study, we report a new generation of ABT-263-based PROTACs designed to leverage a novel solvent-exposed region on the bis(sulfonyl)benzene ring of ABT-263, made accessible through regioselective electrophilic aromatic bromination. The lead compounds, 44 and 46, demonstrated effective degradation of BCL-xL and, unexpectedly, degraded BCL-w, while sparing BCL-2. With further optimization, these BCL-xL and BCL-w dual-targeting PROTACs hold great promise as safer, more effective anticancer agents against BCL-xL and BCL-w codependent cancers.

<i>NF2</i> loss malignantly transforms human pancreatic acinar cells and enhances cell fitness under environmental stress

bioRxiv (Cold Spring Harbor Laboratory) · 2025-05-03

ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) occurs as a complex, multifaceted event driven by the interplay of tumor permissive genetic mutations, nature of cellular origin and microenvironmental stress. In this study, using primary human pancreatic acinar 3D organoids, we performed CRISPR knockout screen targeting 199 previously underappreciated potential tumor suppressors curated from clinical PDAC samples. Our data revealed significant enrichment of a list of candidates, with NF2 emerging as the top target. Functional validation confirmed that loss of NF2 promotes the transition of PDAC to an invasive state, potentially through extracellular matrix modulation. NF2 inactivation was found to enhance PDAC cell fitness under nutrient starvation. This adaptation not only reinforces the oncogenic state but also confers therapeutical resistance. Additionally, we found that NF2 loss is associated with the fibroblast heterogeneity and cancer-stroma communications in tumor evolution. These findings establish NF2 as a critical tumor suppressor in PDAC and uncover its role in mediating nutrient adaptation and drug resistance. Importantly, this study provides new insights into drug resistance mechanisms and potential therapeutic targets in PDAC.

Antibody-Mediated Targeting of Secretory Protein SCUBE3 Suppresses Cancer Progression by Inhibiting Oncogenic Signaling and Inducing Antitumor Immunity

Cancer Research · 2025-12-02

Approaches targeting factors that simultaneously promote tumor growth and progression, induce therapy resistance, and inhibit antitumor immunity offer clear benefits over therapies targeting only one of these tumor-promoting processes. Through comprehensive loss-of-function genomic screening, we identified SCUBE3 as a pivotal factor that supports survival and therapy resistance and also orchestrates an immunosuppressive tumor microenvironment. Secretory SCUBE3 supported oncogenic activity through interactions with key oncogenic cell surface receptor proteins, including EGFR, mutant CALR, and TGFβRI/II. These interactions activated the transcription factors FOXR2 and c-Myc, promoting cancer cell proliferation and therapy resistance by enhancing DNA damage repair. Additionally, the SCUBE3-FOXR2 axis created an immunosuppressive tumor microenvironment by facilitating recruitment of the DNMT1 epigenetic repressor complex to the transcription regulator IRF1, thereby inhibiting the expression of MHC-I and MHC-II genes. A first-in-class neutralizing antibody targeting SCUBE3, which was developed using a sophisticated antibody discovery platform and engineered with specific mutations in the heavy chain for enhanced specificity and efficacy, demonstrated profound therapeutic potential across various cancer types in preclinical models, including patient-derived breast and ovarian cancer xenografts. This discovery marks an advancement toward developing a targeted therapy for cancers characterized by hyperactive SCUBE3-associated signaling pathways. SIGNIFICANCE: Targeting SCUBE3 with a neutralizing antibody inhibits tumor growth and metastasis by blocking oncogenic signaling through FOXR2 and c-Myc and by circumventing immunosuppression, providing a promising pan-cancer treatment approach.

Figure S1 from Targeting DDR2 for Treating Pancreatic Cancer

2025-10-01

&lt;p&gt;Supplementary Figure 1&lt;/p&gt;